The present application is the national stage under 35 U.S.C. 371 of PCT/IL99/00158, filed Mar. 18, 1999.
1. Field of the Invention
The present invention is generally in the field of receptors belonging to the TNF/NGF superfamily of receptors and the control of their biological functions. The TNF/NGF superfamily of receptors includes receptors such as the p55 and p75 tumor necrosis factor receptors (TNF-Rs, hereinafter called p55-R and p75-R) and the FAS ligand receptor (also called FAS/APO1 or FAS-R and hereinafter will be called FAS-R) and others. Specifically, the present invention concerns novel proteins which bind to other proteins which themselves bind directly or indirectly to members of the TNF/NGF receptor family and other intracellular modulatory proteins.
More specifically, it relates to one such protein, herein designated RAP-2 (for RIP-associated protein-2), and its isoforms, fragments, derivatives, and as well as to proteins binding to RAP-2.
RAP-2 binds to RIP (xe2x80x9creceptor interacting proteinxe2x80x9d) and is capable of modulating or mediating the function of RIP and thereby also capable of modulating or mediating, directly or indirectly, the function of other proteins which bind to RIP directly or indirectly. RAP-2 binding proteins are modulators/mediators of RAP-2 function.
2. Background of the Related Art
Tumor Necrosis Factor (TNF-xcex1) and Lymphotoxin (TNF-xcex2) (hereinafter, TNF, refers to both TNF-xcex1 and TNF-xcex2) are multifunctional pro-inflammatory cytokines formed mainly by mononuclear phagocytes, which have many effects on cells (Wallach, D. (1986) In: Interferon 7 (Ion Gresser, ed.), pp. 83-122, Academic Press, London; and Beutler and Cerami (1987). Both TNF-xcex1 and TNF-xcex2 initiate their effects by binding to specific cell surface receptors. Some of the effects are likely to be beneficial to the organism: they may destroy, for example, tumor cells or virus infected cells and augment antibacterial activities of granulocytes. In this way, ELF contributes to the defense of the organism against tumors and infectious agents and contributes to the recovery from injury. Thus, TNF can be used as an anti-tumor agent in which application it binds to its receptors on the surface of tumor cells and thereby initiates the events leading to the death of the tumor cells. TNF can also be used as an anti-infectious agent.
However, both TNF-xcex1 and TNF-xcex2 also have deleterious effects. There is evidence that overproduction of TNF-xcex1 can play a major pathogenic role in several diseases. For example, effects of TNF-xcex1, primarily on the vasculature, are known to be a major cause for symptoms of septic shock (Tracey et al., 1986). In some diseases, TNF may cause excessive loss of weight (cachexia) by suppressing activities of adipocytes and by causing anorexia, and TNF-xcex1 was thus called cachetin. It was also described as a mediator of the damage to tissues in rheumatic diseases (Beutler and Cerami, 1987) and as a major mediator of the damage observed in graft-versus-host reactions (Piquet et al., 1987). In addition, TNF is known to be involved in the process of inflammation and in many other diseases.
Two distinct, independently expressed, receptors, the p55 and p75 TNF-Rs, which bind both TNF-xcex1 and TNF-xcex2 specifically, initiate and/or mediate the above noted biological effects of TNF. These two receptors have structurally dissimilar intracellular domains suggesting that they signal differently (See Hohmann et al., 1989; Engelmann et al., 1990; Brockhaus et al., 1990; Leotscher et al., 1990; Schall et al., 1990; Nophar et al., 1990; Smith et al., 1990; and Heller et al., 1990). However, the cellular mechanisms, for example, the various proteins and possibly other factors, which are involved in the intracellular signaling of the p55 and p75 TNF-Rs have yet to be elucidated. It is this intracellular signaling, which occurs usually after the binding of the ligand, i.e., TNF (xcex1 or xcex2), to the receptor, that is responsible for the commencement of the cascade of reactions that ultimately result in the observed response of the cell to TNF.
As regards the above-mentioned cytocidal effect of TNF, in most cells studied so far, this effect is triggered mainly by the p55 TNF-R. Antibodies against the extracellular domain (ligand binding domain) of the p55 TNF-R can themselves trigger the cytocidal effect (see EP 412486) which correlates with the effectivity of receptor cross-linking by the antibodies, believed to be the first step in the generation of the intracellular signaling process. Further, mutational studies (Brakebusch et al., 1992; Tartaglia et al., 1993) have shown that the biological function of the p55 TNF-R depends on the integrity of its intracellular domain. Accordingly it has been suggested that the initiation of intracellular signaling leading to the cytocidal effect of TNF occurs as a consequence of the association of two or more intracellular domains of the p55 TNF-R. Moreover, TNF (xcex1 and xcex2) occurs as a homotrimer, and as such, has been suggested to induce intracellular signaling via the p55 TNF-R by way of its ability to bind to and to cross-link the receptor molecules, i.e., cause receptor aggregation.
Another member of the TNF/NGF superfamily of receptors is the FAS receptor (FAS-R) which has also been called the FAS antigen, a cell-surface protein expressed in various tissues and sharing homology with a number of cell-surface receptors including TNF-R and NGF-R. The FAS-R mediates cell death in the form of apoptosis (Itoh et al., 1991), and appears to serve as a negative selector of autoreactive T cells, i.e., during maturation of T cells, FAS-R mediates the apoptopic death of T cells recognizing self-antigens. It has also been found that mutations in the FAS-R gene (lpr) cause a lymphoproliferation disorder in mice that resembles the human autoimmune disease systemic lupus erythematosus (SLE) (Watanabe-Fukunaga et al., 1992). The ligand for the FAS-R appears to be a cell-surface associated molecule carried by, amongst others, killer T cells (or cytotoxic T lymphocytesxe2x80x94CTLs), and hence when such CTLs contact cells carrying FAS-R, they are capable of inducing apoptopic cell death of the FAS-R-carrying cells. Further, monoclonal antibodies have been prepared that are specific for FAS-R, these monoclonal antibodies being capable of inducing apoptopic cell death in cells carrying FAS-R, including mouse cells transformed by cDNA encoding human FAS-R (Itoh et al., 1991).
A number of approaches have been made by the applicants (see for example, European Application Nos. EP 186833, EP 308378, EP 398327 and EP 412486) to regulate the deleterious effects of TNF by inhibiting the binding of TNF to its receptors using anti-TNF antibodies or by using soluble TNF receptors to compete with the binding of TNF to the cell surface-bound TNF-Rs. Further, on the basis that TNF-binding to its receptors is required for the TNF-induced cellular effects, approaches by applicants (see for example EP 568925) have been made to modulate the TNF effect by modulating the activity of the TNF-Rs.
Briefly, EP 568925 relates to a method of modulating signal transduction and/or cleavage in TNF-Rs whereby peptides or other molecules may interact either with the receptor itself or with effector proteins interacting with the receptor, thus modulating the normal function of the TNF-Rs. In EP 568925, there is described the construction and characterization of various mutant p55 TNF-Rs, having mutations in the extracellular, transmembrane, and intracellular domains of the p55 TNF-R. In this way, regions within the above domains of the p55 TNF-R were identified as being essential to the functioning of the receptor, i.e., the binding of the ligand (TNF) and the subsequent signal transduction and intracellular signaling which ultimately results in the observed TNF-effect on the cells. Further, there is also described a number of approaches to isolate and identify proteins, peptides or other factors which are capable of binding to the various regions in the above domains of the TNF-R, which proteins, peptides and other factors may be involved in regulating or modulating the activity of the TNF-R. A number of approaches for isolating and cloning the DNA sequences encoding such proteins and peptides; for constructing expression vectors for the production of these proteins and peptides; and for the preparation of antibodies or fragments thereof which interact with the TNF-R or with the above proteins and peptides that bind various regions of the TNF-R., are also set forth in EP 568925. However, EP 568925 does not specify the actual proteins and peptides which bind to the intracellular domains of the TNF-Rs (e.g., p55 TNF-R), nor does it describe the yeast two-hybrid approach to isolate and identify such proteins or peptides which bind to the intracellular domains of TNF-Rs. Similarly, in EP 568925 there is no disclosure of proteins or peptides capable of binding the intracellular domain of FAS-R.
While it is known that the tumor necrosis factor (TNF) receptors, and the structurally-related receptor FAS-R, trigger in cells, upon stimulation by leukocyte-produced ligands, destructive activities that lead to their own demise, the mechanisms of this triggering are still little understood. Mutational studies indicate that in FAS-R and the p55 TNF receptor (p55-R) signaling for cytotoxicity involve distinct regions within their intracellular domains (Brakebusch et al., 1992; Tartaglia et al., 1993; Itoh and Nagata, 1993). These regions (the xe2x80x98death domainsxe2x80x99) have sequence similarity. The xe2x80x98death domainsxe2x80x99 of both FAS-R and p55-R tend to self-associate. Their self-association apparently promotes that receptor aggregation which is necessary for initiation of signaling (see Song et al., 1994; Wallach et al., 1994; Boldin et al., 1995), and at high levels of receptor expression can result in triggering of ligand-independent signaling (Boldin et al., 1995).
Like other receptor-induced effects, cell death induction by the TNF receptors and FAS-R occurs via a series of protein-protein interactions, leading from ligand-receptor binding to the eventual activation of enzymatic effector functions, which in the case studies have elucidated non-enzymatic protein-protein interactions that initiate signaling for cell death: binding of trimeric TNF or the FAS-R ligand molecules to the receptors, the resulting interactions of their intracellular domains (Brakebusch et al., 1992; Tartaglia et al., 1993; Itoh and Nagata, 1993) augmented by a propensity of the death-domain motifs to self-associate (Boldin et al., 1995a), and induced binding of two cytoplasmic proteins (which can also bind to each other) to the receptors"" intracellular domainsxe2x80x94MORT-1 (or FADD) to FAS-R (Boldin et al., 1995b; Chinnaiyan et al., 1995; Kischkel et al., 1995) and TRADD to p55-R (Hsu et al., 1995; Hsu et al., 1996). Three proteins that bind to the intracellular domain of FAS-R and p55-R at the xe2x80x98death domainxe2x80x99 region involved in cell-death induction by the receptors through hetero-association of homologous regions and that independently are also capable of triggering cell death were identified by the yeast two-hybrid screening procedure. One of these is the protein, MORT-1 (Boldin et al. 1995b), also known as FADD (Chinnaiyan et al., 1995) that binds specifically to FAS-R. The second one, TRADD (see also Hsu et al., 1995, 1996), binds to p55-R, and the third, RIP (see also Stanger et al., 1995), binds to both FAS-R and p55-R. Besides their binding to FAS-R and p55-R, these proteins are also capable of binding to each other, which provides for a functional xe2x80x9ccross-talkxe2x80x9d between FAS-R and p55-R. These bindings occur through a conserved sequence motif, the xe2x80x98death domain modulexe2x80x99 common to the receptors and their associated proteins. Furthermore, although in the yeast two-hybrid test MORT-1 was shown to bind spontaneously to FAS-R, in mammalian cells, this binding takes place only after stimulation of the receptor, suggesting that MORT-1 participates in the initiating events of FAS-R signaling. MORT-1 does not contain any sequence motif characteristic of enzymatic activity, and therefore, its ability to trigger cell death does not seem to involve an intrinsic activity of MORT-1 itself, but rather, activation of some other protein(s) that bind MORT-1 and act further downstream in the signaling cascade. Cellular expression of MORT-1 mutants lacking the N-terminal part of the molecule has been shown to block cytotoxicity induction by FAS/APO1 (FAS-R) or p55-R (Hsu et al., 1996; Chinnaiyan et al., 1996), indicating that this N-terminal region transmits the signaling for the cytocidal effect of both receptors through protein-protein interactions.
Thus, the xe2x80x98death domainxe2x80x99 motifs of the receptors p55-R and FAS-R as well as their three associated proteins MORT-1, RIP and TRADD appear to be the sites of protein-protein interactions. The three proteins MORT-1, RIP and TRADD interact with the p55-R and FAS-R intracellular domains by the binding of their death domains to those of the receptors, and for both RIP and TRADD their death domains also self-associate, (although MORT-1 differs in this respect in that its death domain does not self-associate). Further, MORT-1 and TRADD bind differentially to FAS-R and p55-R and also bind to each other. Moreover, both MORT-1 and TRADD bind effectively to RIP. Accordingly, it would seem that the interaction between the three proteins MORT-1, RIP and TRADD is an important part of the overall modulation of the intracellular signaling mediated by these proteins. Interference of the interaction between these three intracellular proteins will result in modulation of the effects caused by this interaction. For example, inhibition of TRADD binding to MORT-1 may modulate the FAS-R-p55 TNF-R interaction. Likewise, inhibition of RIP in addition to the above inhibition of TRADD binding to MORT-1 may further modulate FAS-R-p55 TNF-R interaction.
Monoclonal antibodies raised against the xe2x80x98death domainxe2x80x99 of p55-R, specifically against the binding site of sites of TRADD and RIP can also be used to inhibit or prevent binding of these proteins and thus cause modulation of the interaction between FAS-R and p55-R.
It has also recently been found that besides the above noted cell cytotoxicity activities and modulation thereof mediated by the various receptors and their binding proteins including FAS-R, p55-R, MORT-1, TRADD, RIP, MACH, Mch4, and G1, a number of these receptors and their binding proteins are also involved in the modulation of the activity of the nuclear transcription factor NF-xcexaB, which is a key mediator of cell survival or viability, being responsible for the control of expression of many immune- and inflammatory-response genes. For example, it has been found that TNF-xcex1 can actually stimulate activation of NF-xcexaB and thus TNF-xcex1 is capable of inducing two kinds of signal in cells, one eliciting cell death and another that protects cells against death induction by inducing gene expression via NF-xcexaB (see Beg and Baltimore, 1996; Wang et al., 1996; Van Antwerp et al., 1996). A similar dual effect for FAS-R has also been reported (see reference to this effect as stated in above Van Antwerp et al., 1996). It would therefore appear that there exists a delicate balance between cell death and cell survival upon stimulation of various types of cells with TNF-xcex1 and/or the FAS-R ligand, the ultimate outcome of the stimulation depending on which intracellular pathway is stimulated to a greater extent, the one leading to cell death (usually by apoptosis), or the one leading to cell survival via activation of NF-xcexaB.
In addition, the present inventors have also recently further elucidated the possibly pathway by which members of the TNF/NGF receptor family activate NF-xcexaB (see Malinin et al., 1997 and the various relevant references set forth therein; and co-owned, co-pending Israel Patent Application Nos. IL 117800 and IL 119133). Briefly, it arises that several members of the TNF/NGF receptor family are capable of activating NF-xcexaB through a common adaptor protein, TRAF2. A newly elucidated protein kinase called NIK (see above Malinin et al., 1997 and IL 117800 and IL 119133) is capable of binding to TRAF2 and of stimulating NF-xcexaB activity. In fact, it was shown (see aforesaid Malinin et al. and IL applications) that expression in cells of kinase-deficient NIK mutants results in the cells being incapable of having stimulation of NF-xcexaB in a normal endogenous manner and also in the cell having a block in induction of NF-xcexaB activity by TNF, via either FAS-R, and a block in NF-xcexaB induction by TRADD, RIP and MORT-1 (which are adaptor proteins that bind these p55-R and/or FAS-R receptors). All of the receptors p55-R, p75-R, FAS-R and their adaptor proteins MORT-1, TRADD and RIP bind directly or indirectly to TRAF2, which by its binding ability to NIK apparently modulates the induction of NF-xcexaB.
Of the above modulator proteins involved in the fine balance between cell death and survival following stimulation of FAS-R and/or p55-R, the protein RIP appears to have an important role. RIP (see Stanger et al., 1995 and also Malinin et al., 1997) has a xe2x80x98death domainxe2x80x99 in its C-terminal region which enables it to induce cell cytotoxicity in an independent way and also by association with the death domains of MORT-1, p55-R, FAS-R and TRADD. RIP also has a protein kinase domain at its N-terminal region and an intermediate domain which is believed to enable its intersection (binding) with TRAF2 and thereby its involvement in NF-xcexaB induction. Accordingly, details concerning the characteristics and sequences (DNA and amino acid) of RIP are set forth in the above noted publications (in particular, Stanger et al., 1995) which are incorporated herein in their entirety by reference.
TNF is also one of the cytokines involved in initiation and modulation of the host anti-viral defense. Similarly, viruses have evolved to express genes whose proteins regulate activity of the cytokines, and these cytokine-regulatory viral proteins are thought to promote persistence of the virus within the animal host. One of the best-studied examples of such a protein is E3-14.7K from the group C human adenoviruses (Ad) of types 2 and 5 which acts as a strong antagonist of TNF-mediated cytolysis.
With the aim of isolating molecular components of the TNF signaling cascade that become targets for E3-14.7K upon viral infection, a human E3-14.7K binding protein was recently isolated by two hybrid screening (FIP-2 for Fourteen-K Interacting Protein, Li. Y. et al. 1998). FIP-2 was found to be non-toxic on its own, and to reverse the protective effect of E3-14.7K on cytotoxicity, induced by over-expression of TNFR-1 or RIP, without binding to either of the two above-mentioned proteins. FIP-2 was found to have some homology to RAP-2, the protein of the present invention. The degree of overall similarity between RAP-2 and FIP-2 nevertheless is fairly low, as can be seen from the global alignment of the two amino acid sequences (FIG. 3). The homology however becomes more significant in specific regions towards the C-terminus of the proteins, culminating in virtual identity of the 30 C-terminal amino acids. It is noteworthy that, besides the abovementioned C-terminal domain, the putative Leucine Zipper motif in FIP-2 is largely preserved in RAP-2 (except for an lie to Ala substitution).
A similar sequence named HYPLxe2x80x94encoding a protein related to Huntington""s disease that appears to be a distant homolog of RAP-2 was recently submitted in GenBank under the title xe2x80x9chuntingtin interacting protein, HYPLxe2x80x9d (accession number AF049614). However, a publication describing the function of the protein not yet been published .
A recent publication by Yamaoka S. et al. (1998), reports the identification of a murine RAP-2 homolog. The murine homolog NEMO (for NF-xcexaB Essential Modulator) was identified in a search for the key molecules that regulate the activation of NF-xcexaB signaling. A flat cellular variant of HTLV-I Tax-transformed rat fibroblasts was characterized, denominated 5R, which was unresponsive to all tested NF-xcexaB-activating stimuli (LPS, PMA, IL-I, TNF), and performed its genetic complementation. As a result of this procedure, a cDNA encoding the NEMO 48 kD protein was recovered. Based on this data, this protein is said to be absent from 5R cells, is part of the high molecular weight Ixcexa B-kinase complex, and is requested for its formation. In vitro, NEMO can homo-dimerize and directly interacts with IKKxcex2.
Israel patent specification No. 120485 discloses a RIP-associated protein, termed RAP, which specifically binds to RIP and inhibits NF-xcexaB induction.
Israel patent specificaion No. 123758 and this application relate to another RIP-associated protein termed RAP-2, which has the same or similar activities.
RAP-2 according to the invention is also called 303 or RAP-303 or RAT-303. For consistency""s sake, it will be called RAP-2 herein.
It is an object of the invention to provide a novel protein RAP-2, including all isoforms, analogs, fragments or derivatives thereof, capable of binding to the RIP protein (herein after xe2x80x98RIPxe2x80x99). As RIP is capable of interacting directly or indirectly with the intracellular mediators of inflammation, cell cytotoxicity/cell death, such as p55-R and FAS-R and their associated adaptor or modulator proteins such as, for example, MORT-1, TRADD, MACH, Mch4, G1 and others, the novel proteins of the present invention by binding to RIP are therefore capable of affecting the intracellular signaling process initiated by the binding of the FAS ligand to its receptor, and TNF to its receptor (p55-R), and as such the new proteins of the present invention are modulators of the p55-R and FAS-R-mediated effect on cells. RIP is also capable of interacting with TRAF2 and thereby is capable of interacting directly or indirectly with NIK and as such RIP acts as a modulator of inflammation and of cell survival pathways involving NF-xcexaB induction, thus the new proteins of the present invention are modulators of RIP-related inflammation and cell survival activity. Likewise, by way of the FAS-R, p55-R and their modulator proteins MORT-1 and TRADD being capable of inducing NF-xcexaB and cell survival either directly or indirectly by binding to RIP or by binding to TRAF2, to which RIP binds, the proteins of the present invention may also be mediators of cell survival processes by way of operating via common or related intracellular signaling pathways in which the various above proteins operate to induce cell survival. Similarly, as p75-R binds to TRAF2 to which RIP binds, the novel proteins of the invention may also be modulators of RIP-related mediation of p75-R mediated activity.
Another object of the invention is to provide antagonists (e.g., antibodies, peptides, organic compounds, or even some isoforms) to the above novel RAP-2 proteins, isoforms, analogs, fragments and derivatives thereof, which may be used to inhibit the signaling process, or, more specifically, the inflammation cell-cytotoxicity, or cell-survival processes, when desired.
A further object of the invention is to use the above novel RAP-2 proteins, isoforms, analogs, fragments and derivatives thereof, to isolate and characterize additional proteins or factors, which may be involved in regulation of receptor activity, e.g., other proteins which may bind to RAP-2 proteins and influence their activity, and/or to isolate and identify other receptors further upstream or downstream in the signaling process(es) to which these novel proteins, analogs, fragments and derivatives bind, and hence, in whose function they are also involved.
The invention this also provides RAP-2 binding proteins which are capable of modulating/mediating RAP-2 function.
A still further object of the invention is to provide inhibitors which can be introduced into cells to bind or interact with RAP-2 and possible RAP-2 isoforms which inhibitors may act to inhibit RIP-associated activity in cell cytotoxic processes and hence, when desired, to enhance cell survival, or which may act to inhibit RIP-associated activity in cell-survival processes and hence, when desired, to enhance cell cytotoxicity.
Moreover, it is an object of the present invention to use the above-mentioned novel RAP-2 proteins, isoforms and analogs, fragments and derivatives thereof as antigens for the preparation of polyclonal and/or monoclonal antibodies thereto. The antibodies, in turn, may be used, for example, for the purification of the new proteins from different sources, such as cell extracts or transformed cell lines.
Furthermore, these antibodies may be used for diagnostic purposes, e.g., for identifying disorders related to abnormal functioning of cellular effects mediated by the p55-R, FAS-R or other related receptors.
A further object of the invention is to provide pharmaceutical compositions comprising the above novel RAP-2 proteins, isoforms, or analogs, fragments or derivatives thereof, as well as pharmaceutical compositions comprising the above noted antibodies or other antagonists.
In accordance with the present invention, a novel protein RAP-2 has been isolated. RAP-2 is capable of binding to, or interacting with, RIP, and hence is a modulator or mediator of RIP intracellular activity. RIP is involved in the modulation or mediation of intracellular signaling pathways, e.g. the cell cytotoxicity or cell death associated pathway in which RIP has cytotoxic activity by itself and in association, directly or indirectly, with a number of other cell-death associated proteins, such as, for example, MORT-1, TRADD, MACH, Mch4, G1, p55-R and FAS-R, with which RIP can associate or bind to in a direct or indirect fashion via the xe2x80x98death domainxe2x80x99 motif/module present in RIP and in all the aforesaid proteins; another pathway being the inflammation, cell survival or viability pathway in which RIP may have an activation role, directly or indirectly by virtue of the presence of a kinase motif or domain present in RIP and RIP""s ability to be capable of binding to TRAF2 which can bind NIK which, in turn, is directly involved in activation of NF-xcexaB which plays a central role in inflammation and cell survival. Further, p55-R is also capable of interaction with TRADD and TRAF2 (via TRADD) and is also implicated in NF-xcexaB activation and thereby in the cell survival pathway, and hence RIP by being capable of binding to or interacting with, FAS-R, TRADD and p55-R (via TRADD) as well as with TRAF2 may also be implicated in the modulation of inflammation, cell survival activation by these proteins. Accordingly, RIP is a modulator or mediatior of these pathways, and likewise, the new RAP-2 of the present invention by binding to RIP is a modulator or mediator of these intracellular pathways.
RAP-2 has been isolated and cloned using the yeast two-hybrid system, sequenced and characterized, and as is detailed herein below, RAP-2 appears to be a highly specific RIP-binding protein and hence a specific RIP modulator/mediator. RAP-2 does not bind to TRADD, MORT-1, p55-R, p75-R and MACH. Further, it appears that RAP-2 does not have a characteristic death domain module or motif, this being consistent with the finding that RAP-2 does not induce cell cytotoxicity on its own.
As will be used herein throughout, RIP activity is meant to include its activity in modulation/mediation in the inflammation and cell death/survival pathways. These activities are indicated hereinabove and hereinbelow as well as in all the above-mentioned publications and patent applications, the full contents of which are incoroporated herein by reference. Likewise, as used herein throughout RAP-2 activity is meant to include its modulation/mediation of RIP activity by virtue of its specific binding to RIP, this modulation/mediation of RIP by RAP-2 including modulation/mediation of the inflammation, cell death and cell survival pathways in which RIP is involved directly or indirectly, and as such RAP-2 may be considered as an indirect modulator/mediator of all the above mentioned proteins and possibly a number of others which are involved in inflammation, cell death or cell survival and to which RIP binds, or with which RIP interacts in a direct or indirect fashion.
This invention also discloses two novel RAP-2 binding proteins, identified by two hybrid screening using the full length RAP-2 protein sequence as bait.
Applying the full-length RAP-2 protein as bait in two-hybrid screen a novel RAP-2-interacting protein denoted hereabove or hereafter clone #10 (or clone #10-encoded protein or RAT-binding protein #10 or RBP-10). The sequence of the cDNA obtained was further extended by common sequencing methods known in the art towards the 5xe2x80x2 end, to reconstitute a partial open reading frame of the protein which however lacks a start codon.
Two-hybrid assay of the binding repertoire of clone #10 revealed that this protein, not only binds RAP-2, but exhibits also a rather strong affinity to TRAF2. Clone #10 however does not bind to RIP, TRADD, MORT1, MACH, TNFR-1, TIP60 and NIK as well as to several control proteins (for example lamin and cyclinD). It cannot however be excluded that binding of clone #10 to NIK might be found in mammalian cells, considering the peculiarities of NIK""s behaviour in yeast. Clone #10 was shown to bind RAP-2 within the C-terminal 200 a.a. of the latter, i.e. a region not necessarily associated with the binding of RIP, TIP60, NIK and IKKxcex2. This sequence, however inaccurate, enabled us to carry out several rounds of GenBank searches aiming at identification of homologues of clone #10. The only protein that exhibited a substantial degree of similarity to the protein encoded by Clone #10 was F40F12.5xe2x80x94a hypotetical molecule from C.Elegans, to which no physiological role is assigned.
Interestingly, F40F12.5 was found to display some similarity to several members of the widely conserved family of ubiquitin-directed proteases. These enzymes counterbalance the destructive effect of the ubiquitination machinery, which is known to be in charge of the majority of protein degradation events in a cell. While ubiquitin ligases are responsible for attaching the poly-ubiquitin tree to a protein predestiied for degradation, ubiquitin proteases prevent an effective branching of the growing tree. Such presumption regarding the function of F40F12.5 based on the similarity to the abovementioned ubiquitin-directed proteases however is questionable, as it has not yet been examined whether this particular protein posesses any enzymatic activity toward ubiquitin polymers. Furthermore a couple of points make such a coincidence quite unlikely:
a) Residues which are believed to constitute the core catalytic region in either subclass of ubiquitin proteases are not conserved in either F40F12.5, or in Clone #10;
b) Except from their catalytic sites, enzymes of the ubiquitin-directed protease family derived from various species (from bacteria to human) display virtually no sequence similarity while F40F12.5 and clone #10 dispaly a certain degree of homology.
It thus appears that RAP-2 is a specific RIP-binding protein and hence a modulator/mediator of RIP intracellular activity. The RAP-2 binding proteins, by their ability to bind RAP-2, have indirect influence on RIP and are thus also modulators/mediators of RIP intracellular activity.
Thus, as RAP-2 apparently has a role in modulating/mediating inflammation, cell survival and/or cell death activities in which RIP is involved directly or indirectly, especially those related to cytotoxicity and inflammation caused or induced by various stimuli including those transmitted via receptors of the TNF/NGF receptor family and possibly others as well. (For a scheme of RIP""s involvement in these intracellular events and hence RAP-2""s involvement, see FIG. 1 in Malinin et al., 1997).
RAP-2 may also serve as an inhibitor of cell cytotoxicity and inflammation by virtue of its being present as part of a complex of other proteins, e.g. RIP and proteins bound to RIP, and as such may affect the cytotoxicity or inflammatory effects of these other proteins (e.g. p55-R, FAS-R, MACH, Mch4, G1 and MORT-1), ultimately resulting in an inhibition of their cytotoxic activity or their activity in inflammation.
RAP-2 may yet also serve as an enhancer or augmentor of cell cytotoxicity and inflammation and thus by augmenting the activity of other proteins, e.g. RIP and other proteins bound to RIP as noted above aiding in the recruitment of these proteins by RIP, the recruitment serving to augment the cytotoxic activity of the various proteins or to augment their inflammatory effects.
Likewise, in an analogous fashion RAP-2 may also serve as an inhibitor or an augmentor of the cell-survival pathway as noted above by virtue of RIP""s involvement in this pathway.
Accordingly, the present invention provides a DNA sequence encoding a RIP-associated protein (RAP-2), isoforms, analogs or fragments thereof, capable of binding to RIP and modulating or mediating the intracellular activity of RIP, said intracellular activity being a modulation/mediation of inflammation and/or cell death and/or cell survival.
In particular, the present invention provides a DNA sequence selected from the group consisting of:
(a) a cDNA sequence derived from the coding region of a native RAP-2 protein;
(b) DNA sequences capable of hybridization to a sequence of (a) under moderately stringent conditions and which encode a biologically active RAP-2 protein; and
(c) DNA sequences which are degenerate as a result of the genetic code to the DNA sequences defined in (a) and (b) and which encode a biologically active RAP-2 protein.
Another specific embodiment of the above DNA sequence of the invention is a DNA sequence comprising at least part of the sequence encoding at least one isoform of the RAP-2 protein. Another embodiment of the above DNA sequence is the sequence encoding the RAP-2 protein as depicted in FIG. 1. Yet another embodiment is the DNA sequence shown in FIG. 2.
The present invention provides RAP-2 proteins, and analogs, fragments or derivatives thereof encoded by any of the above sequences of the invention, said proteins, analogs, fragments and derivatives being capable of binding to RIP and modulating/mediating its biological activity in cell death and/or cell survival pathways intracellularly.
A specific embodiment of the invention is the RAP-2 protein, analogs, fragments and derivatives thereof. The RAP-2 protein sequence as deduced from the DNA sequences of FIGS. 1 and 2 is shown in FIG. 3. Another embodiment is any isoform of the RAP-2 protein, analogs, fragments and derivatives thereof.
Also provided by the present invention are replicable expression vehicles comprising the above DNA, these replicable expression vehicles being capable of being expressed in suitable eukaryotic or prokaryotic host cells; transformed eukaryotic or prokaryotic host cells containing such replicable expression vehicles; and a method for producing the RAP-2 protein, or analogs, fragments or derivatives of the invention by growing such transformed host cells under conditions suitable for the expression of said protein, analogs, fragments or derivatives, effecting post-translational modifications of said protein as necessary for obtaining said protein and extracting said expressed protein, analogs, fragments or derivatives from the culture medium of said transformed cells or from cell extracts of said transformed cells. The above definitions are intended to include all isoforms of the RAP-2 protein.
In another aspect, the present invention also provides antibodies or active derivatives, or fragments thereof specific for the RAP-2 protein, and analogs, fragments and derivatives thereof, of the invention.
By yet another aspect of the invention, there are provided various uses of the above DNA sequences or the proteins which they encode, according to the invention, which uses include amongst others:
(i) A method for the modulation of the intracellular inflammation, cell death and/or cell survival pathways modulated or mediated by the protein RIP, comprising treating said cells with one or more RAP-2 proteins, isoforms, analogs, fragments or derivatives thereof, capable of binding to RIP wherein said treating of said cells comprises introducing into said cells said one or more proteins, isoforms, analogs, fragments or derivatives thereof in a form suitable for intracellular introduction thereof, or introducing into said cells a DNA sequence encoding said one or more proteins, isoforms, analogs, fragments or derivatives in the form of a suitable vector carrying said sequence, said vector being capable of effecting the insertion of said sequence into said cells in a way that said sequence is expressed in said cells.
(ii) A method for the modulation of the inflammation, cell death and/or cell survival pathways mediated by ligands of the TNF family by effect on cells via the action of the RIP protein, according to (i) above, wherein said treating of cells comprises introducing into said cells said RAP-2 protein, or isoforms, analogs, fragments or derivatives thereof, in a form suitable for intracellular introduction, or introducing into said cells a DNA sequence encoding said G1 protein, or isoforms, analogs, fragments or derivatives in the form of a suitable vector carrying said sequence, said vector being capable of effecting the insertion of said sequence into said cells in a way that said sequence is expressed in said cells.
(iii) A method as in (ii) above wherein said treating of said cells is by transfection of said cells with a recombinant animal virus vector comprising the steps of:
(a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein (ligand) that is capable of binding to a specific cell surface receptor on the surface of a FAS-R- or p55-R-carrying cell and a second sequence encoding a protein selected from RAP-2 protein, and isoforms, analogs, fragments and derivatives thereof, that when expressed in said cells is capable of modulating/mediating the intracellular inflammation, cell death and/or cell survival pathways; and
(b) infecting said cells with said vector of (a).
(iv) A method for modulating the inflammation, cell death and/or cell survival pathways mediated by the ligands of the TNF family effect on cells via the action of the RIP protein comprising treating said cells with antibodies or active fragments or derivatives thereof, according to the invention, said treating being by application of a suitable composition containing said antibodies, active fragments or derivatives thereof to said cells, wherein when at least part of the RAP-2 protein is exposed on the extracellular surface, said composition is formulated for extracellular application, and when said RAP-2 proteins are entirely intracellular, said composition is formulated for intracellular application.
(v) A method for modulating the inflammation, cell death and/or cell survival pathways mediated by the ligands of the TNF family effect on cells via the action of the RIP protein comprising treating said cells with an oligonucleotide sequence encoding an antisense sequence of at least part of the RAP-2 protein sequence of the invention, said oligonucleotide sequence being capable of blocking the expression of the RAP-2 protein.
(vi) A method as in (ii) above for treating tumor cells or HIV-infected cells or other diseased cells, comprising
(a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein capable of binding to a specific tumor cell surface receptor or HIV-infected cell surface receptor or receptor carried by other diseased cells and a sequence encoding a protein selected from RAP-2 protein, analogs, fragments and derivatives of the invention, that when expressed in said tumor, HIV-infected, or other diseased cell is capable of killing said cell via the action of the RIP protein; and
(b) infecting said tumor or HIV-infected cells or other diseased cells with said vector of (a).
(vii) A method for modulating the cell death and/or cell survival pathways mediated by ligands of the TNF family effect on cells via the action of the RIP protein comprising applying the ribozyme procedure in which a vector encoding a ribozyme sequence capable of interacting with a cellular mRNA sequence encoding a RAP-2 protein according to the invention, is introduced into said cells in a form that permits expression of said ribozyme sequence in said cells, and wherein when said ribozyme sequence is expressed in said cells it interacts with said cellular mRNA sequence and cleaves said mRNA sequence resulting in the inhibition of expression of said RAP-2 protein in said cells.
(viii) A method selected from the above methods according to the invention, wherein said RAP-2 protein encoding sequence comprises at least one of the RAP-2 isoforms, analogs, fragments and derivatives of any thereof according to the invention which are capable of binding to RIP.
(ix) A method for isolating and identifying proteins, according to the invention capable of binding to the RIP protein, comprising applying the yeast two-hybrid procedure in which a sequence encoding said RIP protein or is carried by one hybrid vector and sequence from a cDNA or genomic DNA library is carried by the second hybrid vector, the vectors then being used to transform yeast host cells and the positive transformed cells being isolated, followed by extraction of the said second hybrid vector to obtain a sequence encoding a protein which binds to said RIP protein.
(x) A method according to any of the (i)-(ix) above wherein said RAP-2 protein is any one of the isoforms of RAP-2, analogs, fragments and derivatives of any thereof.
(xi) A method according to any of the above (i)-(x) wherein the RAP-2 protein or any of its isoforms, analogs, fragments or derivatives is involved in the modulation of the cellular effect mediated or modulated by any other mediator or inducer to which said RAP-2 protein, isoform, analog, fragment or derivative is capable of binding directly or indirectly.
The present invention also provides a pharmaceutical composition for the modulation of inflammation, the cell death and/or cell survival pathways mediated by the TNF family effect on cells via the action of the RIP protein or the effect of any other mediator or inducer on cells as noted above, comprising, as active ingredient any one of the following
(i) a RAP-2 protein according to the invention, and biologically active fragments, analogs, derivatives of mixtures thereof;
(ii) a recombinant animal virus vector encoding a protein capable of binding a cell surface receptor and encoding a RAP-2 protein or biologically active fragments or analogs, according to the invention;
(iii) an oligonucleotide sequence encoding an anti-sense sequence of the RAP-2 protein sequence according to the invention, wherein said oligonucleotide may be the second sequence of the recombinant animal virus vector of (ii) above.
The present invention also provides:
I. a method for the modulation of the inflammation, intracellular cell death and/or cell survival pathways modulated/mediated by the RIP protein, or the effect of any other mediator or inducer, or any other NF-xcexaB inducer or inhibitor, on cells comprising treating said cells in accordance with a method of any one of (i)-(x) above, with RAP-2 proteins, isoforms, analogs, fragments or derivatives thereof or with sequences encoding RAP-2 proteins, isoforms, analogs or fragments thereof, said treatment resulting in the enhancement or inhibition of said RIP-mediated effect, and thereby also of the FAS-R or p55-R-mediated effect, or of said other mediator or inducer, or other NF-xcexaB inducer or inhibitor.
II. a method as above wherein said RAP-2 protein, analog, fragment or derivative thereof is that part of the RAP-2 protein which is specifically involved in binding to RIP, or said other mediator or inducer, or other NF-xcexaB inducer or inhibitor, or said RAP-2 protein sequence encodes that part of RAP-2 protein which is specifically involved in binding to RIP, or said other mediator or inducer, or other NF-xcexaB inducer or inhibitor.
III. a method as above wherein said RAP-2 protein is any one of the RAP-2 isoforms, said isoforms capable of enhancing the RIP-associated effect.
IV. a method as above wherein said RAP-2 protein is any one of the RAP-2 isoforms, said isoforms capable of inhibiting the RIP-associated effect, or other mediator or inducer associated effect on cells and thereby also of inhibiting the FAS-R- or p55-R-associated effect on cells, or the other cytotoxic mediator or inducer effect on cells.
V. a method as above wherein said RAP-2 protein, isoform, analog, fragment or derivative capable of enhancing or inhibiting the RIP-associated effect on the inflammation and cell survival pathway by way of direct or indirect inhibition of NF-xcexaB or direct or indirect activation of JNK or p38 kinase.
Isolation of the RAP-2 proteins, their identification and characterization may be carried out by any of the standard screening techniques used for isolating and identifying proteins, for example, the yeast two-hybrid method, affinity chromatography methods, and any of the other well-known standard procedures used for this purpose.
In yet another aspect of the invention, the RAP-2 protein itself, or an isoform, fragment or derivative thereof, is used as bait in a yeast two-hybrid screen for proteins binding thereto.
Proteins which bind to RAP-2, isoforms, fragments or derivatives thereof, are also part of the present invention.
Other aspects and embodiments of the present invention are also provided as arising from the following detailed description of the invention.
It should be noted that, where used throughout, the following terms: xe2x80x9cModulation/Mediation of the RIP, or FAS-ligand, or TNF effect on cellsxe2x80x9d; and any other such xe2x80x9cModulation/Mediationxe2x80x9d mentioned in the specification are understood to encompass in vitro as well as in vivo treatment and, in addition, also to encompass inhibition or enhancement/augmentation.